The long-term goal of this project is to develop and apply the latest proteomics approaches to understand molecular dysfunction underlying the pathogenesis of Alzheimer's disease (AD). Dissection of molecular composition (e.g. DNAs, RNAs, and proteins) in clinical specimens is critical for understanding, detecting, preventing and treating disease. The evolving DNA technology has been revolutionizing medical research and patient care. National Institute on Aging has invested tens of millions of dollars to search for AD risk genes. In contrast, proteomic research is being left behind, largely due to the limitations of technology. Our lab has been developing proteomics approaches for ultra-sensitivity and high throughput for more than a decade. Recently, we have implemented a highly sophisticated platform for profiling more than 10,000 proteins. To explore the proteome alteration in human AD brains, we and collaborators initiated a study to analyze aggregated proteome, revealing unique U1 snRNP pathology in AD and raising a novel concept of RNA splicing alterations in the pathogenesis. Thus, we propose to further enhance the proteomics platform with milestones, and profile AD mouse and human brain proteomes, as well as the interactome of AD disease proteins, for identifying novel pathogenic mechanisms. Three specific aims are: (i) to establish a multiplex, quantitative platform for nearly complete coverage of human proteome as well as comprehensive protein modification analyses, (ii) to identify aberrant protein pathways in AD by profiling the whole proteome and protein modifications in human and mouse models, and (iii) to determine AD disease pathways by the interactome of AD disease proteins in human. Successful outcome of this project will enable truly global proteomic studies of clinical samples, matching the coverage of genomics. The AD study may provide novel targets for pathogenesis and therapeutic intervention.